This invention relates to novel chemiluminescent compounds that are substrates of hydrolytic enzymes, the chemiluminescent products of which have distinctly different light emission characteristics (i.e. emission wavelength, kinetics, or quantum yield). This invention also relates to a light-releasing reagent composition that reacts preferentially with the chemiluminescent substrate or with the chemiluminescent product in the mixture of the two, to generate a discernible signal that can be quantified. This invention further relates to detection methods comprising a novel acridinium-based chemiluminescent substrate, a hydrolytic enzyme and a signal-releasing reagent. This invention furthermore relates to detection devices in conjunction with the use of novel chemiluminescent substrates and hydrolytic enzymes, which include a red-sensitive photomultiplier tube or a charge-coupled device, and a light-filtering device to maximize the detection of chemiluminescent product signal. Further, this invention relates to the use of these novel chemiluminescent substrates in assays to detect, quantitatively or qualitatively, a hydrolytic enzyme of interest that is present either as a label or as a marker of a biological sample. Finally, this invention relates to the process and intermediates for the preparation of these novel chemiluminescent substrates.
The detection of hydrolytic enzymes has been extensively used in diagnostic assays ranging from immunoassays, nucleic acid assays, receptor assays, and other assays, primarily due to their high sensitivity and non-radioactivity. The hydrolytic enzymes include phosphatases, glycosidases, peptidases, proteases, and esterases. By far, most commonly used are phosphatases and glycosidases. For instance, alkaline phosphatase has been extensively used as a label in various enzyme-linked immunosorbent assays (ELISAs) due to its high turn-over rate, excellent thermal stability and ease of use. Many glycosidases, such as xcex2-galactosidase and xcex2-glucuronidase, have also been used in ELISA due to their very high selectivity for the hydrolysis of their preferred substrates. On the other hand, some hydrolytic enzymes play important functions by themselves in biological processes of the human body and microorganisms. Therefore, direct detection of these markers is another important aspect of diagnostics.
In connection with the detection of hydrolytic enzymes, there are three types of substrates: chromogenic, fluorogenic and chemiluminescent substrates. Among them, chemilumi-nescent substrates offer the best enzyme detection sensitivity due to the intrinsic advantages of higher detectability of chemiluminescent product, or lower substrate and instrumental backgrounds, and less interference from biological samples. Therefore, there has been a steady trend towards developing chemiluminescent substrates and applying them in a variety of diagnostics.
One class of widely used chemiluminescent substrates for hydrolytic enzymes are stable dioxetanes (Bronstein et al, U.S. Pat. Nos. 4,931,223, 5,112,960, 5,145,772, 5,326,882; Schaap et al, U.S. Pat. Nos. 5,892,064, 4,959,182, 5,004,565; Akhavan-Tafti et al, U.S. Pat. No. 5,721,370). Here, the thermally stable protective group on the phenolic moiety of the dioxetane substrates is cleaved by a hydrolytic enzyme of interest, such as alkaline phosphatase (AP) or xcex2-galactosidase, depending on whether the protective group is a phosphoryl or xcex2-D-galactopyranosidyl group. The newly generated dioxetane phenoxide anion undergoes auto-decomposition to a methoxycarbonylphenoxide in an electronically excited state. The latter then emits light at xcexmax xcx9c470 nm.
In an aqueous environment where virtually all biological assays are performed, the decomposition of the dioxetanes produces chemiluminescence in a very low quantum yield, typically about 0.01%, and a slow kinetics with t1/2 1xcx9c10 minutes. This is quite different from the decomposition of the dioxetanes in an organic environment. For instance, the dioxetane having the phenol moiety protected by an acetyl group or a silyl group, upon treatment with a base or fluoride, exhibits quantum yield up to 25% in DMSO and 9.4% in acetonitrile, respectively, and t1/2 is about 5 sec at 25xc2x0 C. (Schaap, et al: Tetrahedron Letters, 28 (11), 1155, 1987, and WO 90/07511 A).
Voyta et al (U.S. Pat. No. 5,145,772) disclosed a method of intermolecular enhancement of quantum yield of the dioxetane products using polymeric ammonium salts, which provide a hydrophobic environment for the phenoxide produced by the enzyme.
Akhavan-Tafti et al reported methods of intermolecular enhancement of quantum yield of the dioxetane products using polymeric phosphonium salts (U.S. Pat. No. 5,393,469) and dicationic surfactants (U.S. Pat. Nos. 5,451,347, 5,484,556).
Schaap et al (U.S. Pat. Nos. 4,959,182, 5,004,565) disclosed another method for increasing quantum yield of the dioxetane products using fluorescent co-surfactants as energy acceptors. The resonance energy embodied in the excited phenoxide produced by the enzyme is effectively transferred to the fluorescent co-surfactants. Instead of emitting light at xcexmax 470 nm characteristic of the dioxetane, this system emits light at xcexmax 530 nm as a result of energy transfer to the highly efficient fluorophore, fluorescein.
Another approach for improving quantum yield of the dioxetanes, disclosed by Schaap in U.S. Pat. No. 5,013,827, is to covalently attach a fluorophore having high quantum yield to the light emitting phenoxide moiety. The resonance energy from the excited phenoxide is intramolecularly transferred to the attached fluorophore. The latter in turn emits light at its own wavelength. It is claimed that such dioxetane-fluorophore conjugates exhibit quantum yield as high as 2%.
Wang et al in WO 94/10258 unveiled a class of electron-rich, aryl-substituted dioxetane compounds in which the aryl group is poly-substituted with a suitable electron-donating group so that intense luminescence is observed.
Akhavan-Tafti et al in U.S. Pat. No. 5,721,370 provided a group of stable chemiluminescent dioxetane compounds with improved water solubility and storage stability. The compounds are substituted with two or more hydrophilic groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group.
Schaap et al in U.S. Pat. No. 5,892,064 disclosed a class of chemiluminescent dioxetane compounds substituted on the dioxetane ring with two nonspirofused alkyl groups.
Urdea at al, in EP Application 0401001 A2, described another sub-class of dioxetane compounds that can be triggered by sequential treatment with two different activating enzymes to generate light. The system rests on the principle that the dioxetane substrates have two protecting groups that can are removed sequentially by different processes to produce an excited phenoxide, and the removal of the first protecting group is triggered by the enzyme used as a label in the assay.
Sasamoto at al [Chem. Pharm. Bull., 38(5), 1323 (1990) and Chem. Pharm. Bull., 39(2), 411 (1991)] reported that o-aminophthalhydrazide-N-acetyl-xcex2-D-glucosaminide (Luminol-NAG) and 4xe2x80x2-(6xe2x80x2-diethylaminobenzofuranyl)-phthalhydrazide-N-acetyl-xcex2-D-glucosaminide, both being the non-luminescent forms of luminol, are substrates of N-acetyl-xcex2-D-glucosaminidase. Upon the action of the enzyme on these substrates, luminol or luminol derivative is generated, which then can be detected by triggering with 0.1% hydrogen peroxide and a peroxidase (POD) or no Fe(III)-TCPP complex catalyst to release a chemiluminescent signal.
U.S. Pat. No. 5,306,621 to Kricka disclosed that light intensity of certain peroxidase-catalyzed chemiluminescent reactions can be modulated by AP that acts on a pro-enhancer or a pro-anti-enhancer. For example, the intensity of a chemiluminescent reaction containing luminol, horseradish peroxide and hydrogen peroxide can be enhanced by an enhancer (4-iodophenol) liberated by the enzymatic action of AP on a pro-enhancer (4-iodophenol phosphate), thus enabling AP to be assayed. Alternatively, in the same above reaction where additional enhancer (4-iodophenol) is present, the light intensity can be decreased by an anti-enhancer (4-nitrophenol) generated by the enzymatic action of AP on a pro-anti-enhancer (4-nitrophenol phosphate). In the latter format, the presence of AP can be assayed by measuring the reduction in the light intensity.
Similar to the above, an assay using enzyme-triggerable protected enhancer for quantitation of hydrolytic enzymes was also unveiled by Akhanvan-Tafti in EP Application 0516948 A1.
Akhanvan-Tafti et al in WO/9607911 disclosed another method of detecting hydrolytic enzymes based on the principle of the protected enhancer, where the light emitting species is generated from the oxidation of acridan by peroxidase and peroxide.
Akhanvan-Tafti et al (U.S. Pat. No. 5,772,926; WO 97/26245) disclosed a class of heterocyclic, enol phosphate compounds represented by the non-luminescent acridene enol phosphate. Upon the reaction with a phosphatase enzyme, it is converted to the dephosphoryl enolate, which reacts with molecular oxygen to produce light (see scheme below). It was also disclosed that the light output was greatly enhanced by the addition of a cationic aromatic compound to the assay system. 
Vijay in U.S. Pat. No. 5,589,328 disclosed chemiluminescent assays that detect or quantify hydrolytic enzymes, such as alkaline phosphatase, that catalyze the hydrolysis of indoxyl esters. The assay includes the steps of reacting a test sample with an indoxyl ester and then immediately or within a short time (typically less than about 15 minutes) measuring the resulting chemiluminescence. The resulting chemiluminescence may be amplified by adding a chemiluminescent enhancing reagent.
Among the above chemiluminescent hydrolytic enzyme methods, the dioxetane system appears to be the most sensitive detection system and therefore has been increasingly used in various assays. Despite its widespread use, this system has an inherent drawback in that background chemiluminescence in the absence of enzyme is observed due to slow thermal decomposition and non-enzymatic hydrolysis of the dioxetane. Another intrinsic disadvantage is that the phenoxide, once generated by enzymatic reaction, is extremely unstable and readily undergoes decomposition to release light. In this sense, the phenoxide, the light emitting species, is never xe2x80x9caccumulatedxe2x80x9d during the enzymatic reaction. Therefore, it is one of the major goals of this invention to provide new chemiluminescent substrates whose derived chemiluminescent products have distinguishable emission profiles and whose total signal can be accumulated during the enzymatic reaction, thereby providing an alternative and sensitive detection method for hydrolytic enzymes.
It is an object of this invention to provide chemiluminescent compounds that are substrates of hydrolytic enzymes. Said chemiluminescent substrates upon treatment with a hydrolytic enzyme convert to the corresponding chemiluminescent products that have distinctly different light emission characteristics.
It is another object of this invention to provide acridinium-based chemiluminescent compounds that are substrates of hydrolytic enzymes. Said chemiluminescent substrates contain a phenolic moiety or enol moiety in the molecule that is masked by a group, which is thermally and hydrolytically stable. Upon treatment with a hydrolytic enzyme said chemiluminescent substrates convert to the corresponding acridinium-based chemiluminescent products that have distinctly different light emission characteristics.
It is also an object of this invention that said chemiluminescent substrates and products have distinctly different light emission characteristics, thereby allowing the separation or distinction of the signal of the substrate from the signal of the product or vice versa when both substrate and product are present in the same test vessel.
It is another object of this invention that said chemiluminescent products generated by hydrolytic enzymes have emission maxima different from those of their corresponding chemiluminescent substrates.
It is another object of this invention that said chemiluminescent products generated by hydrolytic enzymes have quantum yields different from those of their corresponding chemiluminescent substrates.
It is another object of this invention that said chemiluminescent products generated by hydrolytic enzymes have light-emitting kinetics different from those of their corresponding chemiluminescent substrates.
It is another object of this invention that said chemiluminescent products generated by hydrolytic enzymes have physical and chemical properties different from those of their corresponding chemiluminescent substrates. Said physical and chemical properties include, but are not limited to, the fundamental net charge distribution, dipole moment, xcfx80-bond orders, free energy, or the apparent hydrophobicity/hydrophilicity, solubility, affinity and other properties which are otherwise apparent to those who are skilled in the art.
It is another object of this invention that said chemiluminescent substrates are structurally manipulated such that the distinction of the light emission characteristics can be further enlarged.
It is yet another object of this invention that chemiluminescent products resulting from the action of hydrolytic enzymes on chemiluminescent substrates do not undergo substantial decomposition during the enzymatic reaction, and thus can be accumulated until triggered by a light-releasing reagent.
The combined objects and advantages of this invention indicated above are attained by:
A. Firstly, a chemiluminescent substrate of hydrolytic enzyme having the following general Formula I, as follows:
xe2x80x83Lumi-M-Pxe2x80x83xe2x80x83Formula I
where xe2x80x9cLumixe2x80x9d is a chemiluminescent moiety capable of producing light (a) by itself, (b) with MP attached and (c) with M attached. Lumi includes, but is not limited to, chemiluminescent acridinium compounds (e.g. acridinium esters, acridinium carboxyamides, acridinium thioesters and acridinium oxime esters) benzacridinium compounds, quinolinium compounds, isoquinolinium compounds, phenanthridinium compounds, and lucigenin compounds, or the reduced (e.g., acridans) or non-N-alkylated forms (e.g., acridines) of the above, spiroacridan compounds, luminol compounds and isoluminol compounds and the like. M is a multivalent heteroatom having at least one lone pair of electrons selected from oxygen, nitrogen and sulfur, directly attached to the light emitting moiety of Lumi at one end and to P at the other end. (When M alone is attached to Lumi to form Lumi-M, it does, of course, have either a proton or a counterion associated with it or is in the form of an ion.). P is a group that can be readily removed by hydrolytic enzymes, as discussed in more detail hereinafter. The light emitting moiety of Lumi is well known.
For example, when Lumi is an acridinium compound or luminol, the light emitting moiety is the acridinium nucleus or phthaloyl moiety, respectively.
B. Secondly, an enzymatic reaction having the following general reaction A, as follows: 
where HE is a hydrolytic enzyme, such as phosphatase, glycosidase, peptidase, protease, esterase, sulfatase and guanidinobenzoatase. Lumi-M-P is a chemiluminescent substrate of a hydrolytic enzyme. Lumi-M is a chemiluminescent product having physical and/or chemical properties different from those of Lumi-M-P. Said physical and/or chemical properties include emission wavelength, quantum yield, light emission kinetics, fundamental net charge distribution, dipole moment, n-bond orders, free energy, or apparent hydrophobicity/hydrophilicity, solubility, affinity and other properties.
C. Light releasing reactions which take place on both Lumi-M-P and Lumi-M.
It is one object of this invention to provide novel light-releasing reagent compositions and reagent addition protocols for triggering light emission from chemiluminescent substrates and products that surprisingly result in better distinction between the signals of the chemiluminescent substrates and products. Said light-releasing reagent compositions can be a single reagent and/or multiple reagents, and addition of said multiple reagents to the reaction vessel can be synchronous or sequential. According to the invention, the light releasing reactions may take place on both Lumi-M-P and Lumi-M, as shown in Formula I.
It is another object of this invention that said light-releasing reagent compositions comprise one or more peroxides or peroxide equivalents, and said peroxides or peroxide equivalents include, but are not limited to, hydrogen peroxide.
It is yet another object of this invention that said light-releasing reagent compositions interact with said chemiluminescent substrate and product differentially so that the differentiation between the two signals is optimized.
It is yet another object of this invention that said light-releasing reagent compositions contain one or more enhancers selected from organic, inorganic or polymeric compounds with a broad range of molecular weights, which differentially enhance the light output from either the substrate or the product.
It is yet another object of this invention that said light-releasing reagent compositions contain also one or more quenchers, blockers or inhibitors selected from organic, inorganic or polymeric compounds with a broad range of molecular weights such that they differentially quench, block or reduce the light output from either the substrate or the product.
The combined objects and advantages of this invention indicated above are attained by a light-releasing reagent composition. Said light releasing reagent composition consists of two separate reagents, which are sequentially added to a solution containing the chemiluminescent substrate and/or product. The first reagent contains acidic hydrogen peroxide solution, and the second reagent contains an alkaline solution with one or more detergents. Alternatively, to the advantage of better distinction between the signals of certain chemiluminescent substrates and their products, the first reagent contains an alkaline solution with one or more detergents, and the second reagent contains hydrogen peroxide solution.
It is another object of this invention to provide optimal light detection methods for said chemiluminescent reactions, so that the differentiation between light emissions of the substrate and product is optimized for specific applications. Said optimal detection methods comprise the use of a light detection apparatus, which includes a luminometer, a Charge-Coupled Device (CCD) camera, an X-ray film, and a high speed photographic film. Said luminometer comprises a blue-sensitive photomultiplier tube (PMT), or a red-sensitive PMT, or other PMTs optimized for specific applications. Said optimal light detection methods also include the use of an optical filtering device to block or reduce unwanted light emission either from the substrate or from the product. Said optimal detection methods further include a method and/or a device for detecting or registering light in a sequential manner that eliminates or reduces unwanted signal from the substrate.
The combined objects and advantages of this invention indicated above are attained by one or more light detection methods. One preferred light detection methods comprises the use of a PMT and a long wave pass filter in an enzymatic reaction to detect the chemiluminescent signal from the product that emits light at a wavelength longer than that of the substrate. Another said light detection method comprises the use of a red-sensitive PMT at low temperature (i.e. below 4xc2x0 C.) and a long wave pass filter to improve the detectability of the chemiluminescent product that emits light at a wavelength longer than that of the substrate. Still, another said light detection method comprises the use of blue-sensitive PMT and a short wave pass filter to detect the decrease of chemiluminescent signal from the substrate whose emission wavelength is shorter than that of the product. Yet, another said light detection method comprises the use of a PMT with or without an optical filter within a fixed light measuring time to detect the decrease of chemiluminescent signal from the substrate whose emission kinetics is faster than that of the product.
It is yet another object of this invention to provide methods and assays comprising the use of one or more said chemiluminescent substrates from Formula I, one or more said hydrolytic enzymes, one or more said light-releasing reagent compositions, and one or more said optimal light detection methods.
It is yet another object of this invention to provide methods and assays in using one or more said chemiluminescent substrates to detect, qualitatively or quantitatively, the presence of one or more said hydrolytic enzymes or enzyme conjugates that are present either as labels or as-markers of biological samples.
It is a further object of this invention to provide method that utilize one or more said chemiluminescent substrates from Formula I, and one or more said labeled hydrolytic enzymes to detect, qualitatively and/or quantitatively, the presence of one or more analytes.
Finally, it is also an object of this invention to provide synthetic methods and intermediates related to the syntheses of said chemiluminescent substrates.